Prion diseases are human and animal conditions which include inherited and sporadic forms as well as a form acquired by infection. Major advances have been made toward proving the "protein only" or prion hypothesis which proposes a general pathogenic mechanism for all prion diseases. However, the precise events involved in the pathogenesis of the individual prion diseases are unknown. To study pathogenesis in detail, we have chosen to focus on the inherited prion diseases, since they can be easily modeled. The pathogenesis of the inherited prion diseases can be viewed as a three stage process: 1) the specific effects of the mutations on the metabolism of the prion protein (PrP), 2) the conversion of the mutant PrP (PrPM) into the PrP isoform that is resistant to proteolytic digestion (Prpres), and 3) the pathogenic effects of the Prprns form. This Program Project focuses on the pathogenesis of prion diseases, and the three Research Projects proposed deal with these stages. In Project 1, we will generate a transgenic mouse model of fatal familial insomnia and a subtype of inherited Creutzfeldt-Jakob disease, two conditions that we have extensively studied in transfected cell models. The metabolism of PrPM will be analyzed in tissue and primary cell cultures obtained from various brain regions of the transgenic mice before and after scrapie infection to transform PrPM into the PrPres form. Proiect 2 deals with the study of the metabolism of PrPM and PrPres in fibroblasts from patients with inherited prion diseases and in differentiated neuronal cells of the human central nervous system (CNS) transfected with the corresponding PrP gene mutant constructs to gain insight into the PrPM metabolism as it occurs in the patient's CNS neurons. Moreover, we will examine the mechanisms of PrPres propagation from cell to cell and from one brain region to another. In Proiect 3, we will characterize PrPres and PrPres fragments by sequence analysis, mass spectrometry, circular dichroism and Fourier transform infrared spectrnscopy in order to identifv mutation-specific covalent modifications as well as secondarv structures. In addition, the conversion of human PrPM into PrPres will be modeled in a cell free system. An Administration Core and a Neuropathology Core are also proposed. The Neuropathology Core will examine the histopathology and immunocytochemistry of transgemic mice generated in Project 1. The investligators participating in this application form a cohesive group with diversified skills. This multidisciplinary study of the pathogenesis of inherited prion diseases offers a unique opportunity to acquire the understanding of these diseases necessary to design of a rational treatment.